PROJECT SUMMARY Focal defects in articular cartilage inevitably lead to post traumatic osteoarthritis, a painful and debilitating disease. A major barrier to the clinical use of adult mesenchymal stem cells (MSCs) in regenerative strategies to repair focal cartilage defects is their limited capacity to produce and retain the structural macromolecules found in healthy articular cartilage tissue that are vital to supporting physiologic loads in synovial joints. This limitation is due, in part, to the activity of transcription factors that regulate the RUNX2 pathway in chondrocytes, resulting in cessation of the production of articular cartilage structural macromolecule by MSC-derived chondrocytes (henceforth called MdChs) and upregulated expression of the proteinases that degrade them. We have designed gene circuits that successfully establishes a synthetic negative feedback loop that inhibits translation of Runx2 in response to increasing levels of Runx2 protein; thereby, maintaining the intracellular Runx2 concentration at low levels. This feedback loop results in decreased expression of proteinases and increased matrix accumulation in mouse stem cell lines. The central objective of this proposal is to use a similar strategy to engineer closed-loop, autoregulatory gene circuits that will enhance the accumulation of cartilage structural macromolecules in human MdChs by suppressing the translation of transcription factors that mediate the effects of RUNX2. The feasibility of this approach to regulate the phenotype of human MdChs will be investigated by rigorously evaluating their response to variation in 2 design parameters of the gene circuit: 1) the choice in transcription factor target?RUNX2 or transcription factors that work synergistically with or upstream of RUNX2; and 2) the sensitivity of circuits to the targeted transcription factor. We will test 2 hypotheses: 1) that the closed-loop synthetic gene circuit proposed here will program genetically modified hMdChs to produce a cartilage tissue with a higher content of cartilage structural macromolecules (Aim 1); and 2) that the gene circuits produced in this proposal will afford protection to programed hMdChs from the effects of inflammatory stimuli that have been shown to induce the activation of RUNX2 signaling in chondrocytes (Aim 2). Accumulation of cartilage structural macromolecules by genetically modified human MdChs will be compared to unmodified MdChs and the current gold standard, healthy articular chondrocytes from aged-matched donors. Most importantly, we will determine the degree to which our gene circuit can produce the desired clinically relevant outcome: generating mechanically competent tissue from programed hMdChs. The outcomes of this work will serve as preliminary data with which we will generate hypotheses about how patient-specific metrics will affect the efficacy of the gene circuit in improving cartilage tissue engineering outcomes.